Determination of cPSA

ABSTRACT

A method for determining the complexed forms of immunologically determinable prostate specific antigen (cPSA) in a blood sample, e.g., by two-site immunometric assays, in which the blood sample is treated to render free PSA (fPSA)immunologically nondetectable. A particularly preferred immunometric assay method employs three anti-PSA antibodies: an antibody that binds to both cPSA and fPSA (anti-tPSA), a second anti-tPSA antibody which is characterized by the unique property that binding to fPSA is blocked by binding of fPSA-specific antibodies, and a third antibody which is a fPSA-specific antibody. Thus, binding of the fPSA-specific antibody to PSA in the sample allows only cPSA to be measured in the immunometric assay. Measurement of cPSA blood levels has been found to provide a method for aiding in the diagnosis and monitoring of prostate cancer that is highly sensitive and specific, and eliminates the need for a significant number of patients to undergo unnecessary prostate biopsy.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of application Ser. No. 08/834,969pending, filed Apr. 7, 1997, which is a continuation-in-part ofapplication Ser. No. 08/738,383, filed Oct. 25, 1996.

BACKGROUND OF THE INVENTION

The present invention relates to the determination of the complexedforms of immunologically determinable prostate specific antigen (PSA) ina blood sample. More particularly, the invention relates to thedetermination of complexed PSA by two-site immunometric assay and theclinical significance of complexed PSA assay values.

Human prostate specific antigen (PSA) is a glycoprotein of approximately33,000 daltons with high amino acid homology to the human kallikreinfamily (1,2) and has been shown to be a serine protease with trypsin andchymotrypsin-like activity (3,4,5). PSA is secreted by epithelial cellsof the prostate gland and is one of the major proteins found in seminalfluid (6). Following the discovery that the concentration of PSAincreases in the serum of patients with prostate cancer, numerousreports have established this protein as an important and clinicallyuseful biomarker for the management of prostate cancer patients(7,8,9,10). Recent efforts have focused on the use of serum PSA testingfor early detection of prostate cancer in asymptomatic men. In fact, theAmerican Cancer Society and the American Urological Society haverecently recommended that all men over the age of 50 be screenedannually using serum PSA in conjunction with digital rectal examination(DRE) (11).

The clinical value of early detection of prostate cancer remainscontroversial for several reasons. First, it is unclear whethertreatment of prostate cancer at early stages will improve survival inthe affected population. Clinical trials designed to address this issueare currently underway. Second, a clinical trial recently measured theeffectiveness of serum PSA measurements in conjunction with digitalrectal examination (DRE) for early detection of prostate cancer in menover 50 years of age (12). Of the 1060 patients who had either anabnormal DRE or an elevated PSA test, only 22% had prostate cancer.These data demonstrate that 70-80% of all prostate biopsies areperformed on men who do not have cancer. Since 30-50% of men over theage of 50 have evidence of prostate cancer on autopsy, the number ofunnecessary prostate biopsies triggered by elevated PSA assays could bevery high. This has consequences both in increased medical costs andincreased morbidity associated with the biopsy procedure.

Several laboratories have shown independently that PSA forms complexeswith protease inhibitors such as α₁ -antichymotrypsin (ACT), α₂-macroglobulin, and α₁ -antitrypsin (13-19). PSA in complex with ACT orα₁ -antitrypsin or in free,uncomplexed form is detectable in serum byimmunoassay techniques. Indeed, the majority of immunoreactive PSA inserum is complexed with ACT, and a significant correlation has beenestablished between the proportion of PSA bound to ACT and total serumPSA concentration (13). PSA bound to α₂ -macroglobulin, however, is notmeasurable in serum due to steric hindrance of antibody binding to PSAfollowing complexation with this protease. In early work, PSA-ACT levelsand the proportion of PSA-ACT to total PSA were suggested to be of usein prostate cancer diagnosis (13,15,16,17), however, for a variety ofreasons (some of which are discussed below) it has been difficult todraw conclusions on the clinical utility of serum measurement ofPSA-ACT.

Lilja, Stenman, and coworkers published in 1991 that serum PSA exists infree form and in complexes with ACT and α₁ -antitrypsin (13,18). Insubsequent work, Stenman et al. demonstrated that measurement of PSA-ACTin association with measurement of free plus complexed PSA (termed totalPSA, although PSA complexed with α₁ -macroglobulin is not measured byconventional PSA assays) may improve discrimination between men withprostate cancer and those with benign prostate disease such as benignprostatic hypertrophy (BPH). However, the accurate measurement ofPSA-ACT complexes has not been attainable due to technical problems inaccurate measurement of the complex. Stenman et al. found that thecorrelation of PSA-ACT values with total PSA measurement was not good atthe low end and the y intercept was elevated indicating over-recovery ofcomplexed PSA (13 and U.S. Pat. No. 5,501,983). Indeed, they found thatfor most patients tested, the concentration of PSA-ACT was higher thanfor total PSA (U.S. Pat. No. 5,501,983). Subsequent correlation analysisfor complexed and free PSA showed a slope of 1.12 indicatingover-recovery of the PSA-ACT complex (16). Pettersson et al. addressedthis over-recovery when they found elevated PSA-ACT values in femalesera (20). While the addition of heparin reduced the incidence of falsepositive values in female serum, more recent attempts to measure PSA-ACTcomplexes in patients with prostate cancer and BPH continue to showsignificant over-recovery of complexes (21).

Because of the difficulties encountered in the measurement of PSA-ACTcomplexes, attention in the literature turned to the measurement offree, uncomplexed PSA in conjunction with measurement of total PSA. Itis now clear that improved specificity is needed when total PSA valuesrange from about 4-10 ng/mL. When serum total PSA is <4.0 ng/mL, therisk of prostate cancer is low; similarly, when total PSA is >10 ng/mL,the risk of prostate cancer is >50% and prostate biopsy is indicated.Within the diagnostic gray zone (generally between 2-20 ng/mL, morecommonly between 4-10 ng/mL) the risk of cancer is high, but the rate offalse positives is also high. The retrospective application of a ratioof free PSA/total PSA has shown that the specificity of total PSA in the4-10 ng/mL gray zone could be improved from approximately 50-60% to70-80% (22-26). This improved specificity could result in a 20-30%decrease in unnecessary biopsies. PCT WO 96-26441 and WO 97-12245similarly describe the use of the free PSA/total PSA ratio to improvediscrimination between BPH and cancer, respectively, in patients withtotal PSA levels between 2.5 and 20 ng/mL.

The measurement of free PSA has technical difficulties of its own,however. First, within the diagnostic gray zone, the proportion of freePSA is typically quite low, in the 5-30% range. A successful free PSAassay must, therefore, measure accurately in the range of 0.2∝3.0 ng/mL.Also, the concentration of free PSA is not significantly different inpatients with BPH and cancer, and the ratio of free PSA/total PSAdecreases due to an increase in the proportion of PSA complexed to ACT.In addition, free PSA is not stable in serum and levels of free PSA havebeen known to decrease over time, presumably due to complexation with α₂-macroglobulin.

In the meantime, there has been further acknowledgment of the problemsassociated with the accurate measurement of PSA-ACT in blood, coupledwith attempts to overcome such problems. In 1994, workers at Hybritechreported the development of a sandwich immunoassay for PSA-ACT employinganti-PSA and anti-ACT antibodies. They concluded that the measuredPSA-ACT values failed to demonstrate improved clinical specificity inthe diagnosis of prostate cancer (27). Later, this group jointly withworkers at the Johns Hopkins Medical Institutions reported the findingthat the anti-PSA/anti-ACT sandwich inmmunoassay method suffers fromsignificant non-specific binding and over recovery of PSA-ACT. Unlessresolved, they concluded that these problems rendered the measurement ofPSA-ACT clinically meaningless (28). Subsequently, this joint groupreported having overcome the non-specific binding problem through thedevelopment of a sandwich immunoassay for PSA-ACT based on a monoclonalantibody specific for PSA-ACT complex (29, 30). However, their clinicalstudies failed to show any improvement in specificity for prostatecancer by measuring PSA-ACT complex alone compared with measurement oftotal PSA or with a calculated ratio of PSA-ACT to total PSA (29). Otherapproaches to overcoming the problems associated with PSA-ACTmeasurement have been proposed, including the use of blocking agents(31).

It remains unclear why the proportion of PSA complexed to ACT increasesin patients with prostate cancer, but it may be related to theobservation that antibodies to ACT do not stain prostatic epitheliumfrom BPH patients and MRNA transcripts are not found in such tissue. Incontrast, anti-ACT immunoreactivity and mRNA synthesis are detected inprostatic epithelium from patients with prostate cancer (32). Theseresults suggest that in prostate tumors, PSA may complex in situ withACT prior to release into serum. An alternative mechanism may involvethe access of active PSA to the blood stream. Free PSA found in serumfrom healthy men is proteolytically cleaved and enzymatically inactive.Tumors, however, synthesize angiogenic factors which lead to increasedvascularization of tumor tissues. It may be that in tumors, a largerproportion of enzymatically active PSA gains access to the blood stream.This active PSA would be expected to complex with protease inhibitorssuch as ACT leading to a higher proportion of PSA-ACT complex in serumfrom prostate cancer patients.

Accordingly, there is a need for an accurate method of determiningcomplexed PSA and to assess the clinical significance of blood levels ofcomplexed PSA relative to screening of male patients for prostatecancer.

EP 635,575 describes the preparation of monoclonal antibodies that bindto free PSA but not PSA-ACT.

PCT WO 95/18381 relates to a monoclonal/polyclonal immunometric assaymethod for the determination of PSA which is rendered capable ofproviding an equimolar response to free and complexed PSA by theaddition of antibody that binds to free PSA but not complexed PSA.

U.S. Pat. application Ser. No. 08/595,155 now abandoned, and Zhou Z, NgPC, Very DL, Allard W J, Yeung K K, J. Clin. Lab. Anal. (1996),10:155-159, describe a method for preparing a monoclonal antibody thatprovides an equimolar response to free and complexed PSA in amonoclonal/polyclonal immunometric assay. The described monoclonalantibody has the unique property of binding to PSA to render PSAsubstantially incapable of binding with antibodies that bind free PSAbut not complexed PSA.

Published Japanese Patent Document 62-46263 describes a sandwichimmunoassay method for the determination of PSA in complex with proteaseinhibitor.

Published German Patent Application 4,322,342 describes a method formeasuring both total PSA and PSA-ACT in a single assay for the purposeof providing values for calculation of the ratio of PSA-ACT to totalPSA.

Chichibu et al, in the Journal of Medicine and Pharmaceutical Science(Japan, 1996) 36(3): 477-483, describe a sandwich immunoassay forPSA-ACT employing anti-PSA bound to a bead and enzyme-labeled anti-ACT.Data establishing the ability to accurately measure PSA-ACT in a bloodsample is lacking.

SUMMARY OF THE INVENTION

The present invention provides a method for determining complexed PSA(herein referred to as cPSA) in a blood sample by treating the bloodsample to render uncomplexed, i.e., free, PSA (fPSA) nondetectable byimmunoassay, and then determining PSA in the treated blood sample byimmunoassay whereby only cPSA is detectable. The immunoassay can beperformed in any conventional manner, but more usually is a competitiveimmunoassay or a two-site immunometric assay. The present method can beaccomplished in a variety of ways as described in more detail below. Ingeneral, such methods include separation methods in which fPSA isphysically removed or retained from the immunoassay test mixture, aswell as methods in which an antigenic determinant or determinants infPSA are modified, such as by chemical interaction, to render fPSAessentially unable to bind to antibody used in the immnunoassay method,thereby effectively eliminating FPSA from the assay.

A particularly advantageous two-site immunometric assay method has beendevised based on a three antibody reagent system:

(a) a first anti-PSA antibody (monoclonal or polyclonal) which binds totPSA and which participates in the immunometric assay,

(b) a second anti-PSA antibody (preferably monoclonal) which also bindsto tPSA and which also participates in the immunometric assay, but whichis selected to have the property that it is substantially incapable ofbinding to PSA when PSA is bound by a fPSA-specific antibody (thissecond antibody is referred to herein at times as "MM1"), and

(c) a third anti-PSA antibody which is fPSA-specific, and preferably ismonoclonal.

As participants in the immunometric assay, one of the first and secondanti-PSA antibodies is labeled for detection purposes (and can bereferred to as the "labeled" or "detection" antibody) and the other isimmobilized or is capable of being immobilized for purposes ofseparation from the test mixture (the "capture" antibody). Accordingly,assay conditions can be established under which FPSA in a blood samplewill bind with the fPSA-specific (third) antibody, rendering fPSA fromthe sample incapable of binding with the aforesaid MM1 (second)antibody. Since the two-site immunometric assay is dependent upon thebinding of both of the aforesaid first and second antibodies (the"labeled" and "capture" antibodies) to PSA, binding of the fPSA-specificantibody consequently renders the fPSA form incapable of detection bythe two-site immunometric assay. It will be noted that despite the factthat the three antibodies used in this particularly unique assay systemare all specific for one or more forms of PSA (that is, none aredirected to any of the protease inhibitors comprised in cPSA), theparticular properties of the antibodies permit the specificdetermination of cPSA.

It has been found that the measurement of cPSA blood levels provides ahighly sensitive and specific method for detecting prostate cancer(CaP). cPSA assays also have the advantage of increased analyticalaccuracy compared to assays involving the measurement of fPSA since cPSAis the predominant form of PSA and environmental and analytical factors(e.g., sample age) affecting the distribution of PSA between the fPSAand cPSA forms produce a much lower effect on the accuracy of cPSAmeasurements by comparison with measurements of fPSA.

Since cPSA is comprised primarily of PSA complexed with the proteaseinhibitor α₁ -antichymotrypsin (ACT), PSA-ACT-specific assays will alsoyield the advantageous sensitivity, specificity, and other features ofthe cPSA assays.

BRIEF DESCRIION OF THE DRAWINGS

FIG. 1A is a graph showing the inhibition of fPSA immunoreactivity in atwo-site immunometric assay for tPSA in the presence of three differentmonoclonal antibodies to the E-epitope of PSA. Samples containing fPSAat 50 ng/mnL were preincubated with each anti-fPSA MAb for 30-60 minutesand run in the Bayer Immuno 1™ tPSA Assay. FIG. 1B shows a similarexperiment in which two anti-E antibodies, PSA 20 and ME2, showed aconcentration dependent inhibition of immunoreactivity of FPSA in thetPSA assay. Samples containing 10 ng/mL fPSA were preincubated witheither the PSA 20 or ME2 MAb for 30-60 minutes and run in the BayerImmuno 1 tPSA Assay.

FIG. 2A is a graph showing that the addition of the anti-E antibody PSA20 to the tPSA assay provides an immunoassay format for the measurementof cPSA. Samples containing approximately 11 ng/mL total PSA withvarying proportions of free and complexed PSA were measured in thepresence of 300 μg/ml MAb PSA 20 using the Bayer Immuno 1 analyzer. FIG.2B is a similar graph showing that ME2 MAb can also be used to providean immunoassay format for the quantitative measurement of cPSA. Samplescontaining approximately 11 ng/ml total PSA with varying proportions offree and complexed PSA were measured in the presence of 25 μg/mL MAb ME2using the Bayer Immuno 1 analyzer.

FIG. 3 is a table showing that PSA 20 can be used to automate the cPSAassay on an automated immunoanalyzer. Assay format 1 used MAb PSA 20added to the MM1-fluorescein conjugate (R1) with a total incubation timeof 38 minutes. Assay format 2 used an on-board preincubation of PSA MAb20 with the samples and a total incubation time of 78 minutes. Allresults are presented as the rate of color formation.

FIGS. 4A and 4B are tables summarizing results of measurement of total,free, and complexed PSA in the serum of men with prostate cancer, BPH orin healthy age-matched controls. The unselected patient populationdesignated "All" includes patient samples derived from men with CaP,BPH, or healthy age-matched controls without regard to tPSA values. Whenpatient groups were stratified according to tPSA value, additionalpatient samples were included in the analysis shown in FIG. 4A and inthe cPSA portion of the analysis shown in FIG. 4B, which additionalsamples were selected by tPSA values for inclusion in the diagnosticgray zone as described in the specification below.

FIG. 4C is a graph of a regression analysis of results obtained using acommercial assay for total PSA compared to results obtained using thepreferred cPSA assay for patient samples collected from men withprostate cancer and benign prostate disease.

FIGS. 5A-5F are graphs showing the correlation between cPSA assay valuesand PSA-ACT assay values obtained from the testing of sera of men withcancer, BPH and in the normal population.

FIG. 6 is a table summarizing results of measurement of cPSA and PSA-ACTin the serum of men with prostate cancer, BPH or in healthy age-matchedcontrols. The unselected population (designated "All") and the patientgroups stratified according to tPSA level were identical to those usedin the studies summarized in FIGS. 4A and 4B.

FIG. 7 is a table showing correlations of cut-off values (i.e., upperlimits of normal) and specificity at selected sensitivities among assayvalues obtained by a commercial tPSA assay, by a preferred cPSA assay,and by calculation of fpSA/tPSA ratios.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the following terms shall have the indicated meanings:

PSA shall mean prostate specific antigen. tPSA or total PSA shall meanthe total amount of immunologically determinable PSA in a blood sample,that is, PSA in complexed or free forms that are capable of respondingto measurement by conventional immunoassays. Based on current knowledge,it is understood that blood PSA that is complexed with certain proteaseinhibitors (including ACT, α₁ -antitrypsin, and inter-α trypsininhibitor) is immunologically determinable, whereas PSA is notdeterminable when complexed with such other protease inhibitors as α₂-macroglobulin.

fPSA or free PSA shall mean PSA in its free, uncomplexed form.

cPSA or complexed PSA shall mean tPSA that is not fPSA.

E-epitope shall mean the collection of epitopes on PSA which are bindingsites for antibodies which bind to fPSA but not to cPSA.

Anti-E antibodies shall mean antibodies which bind to E-epitope, andthus are specific for binding fPSA.

Antibody shall mean whole immunoglobulin, e.g., IgG or IgM, or animmunoglobulin fragment comprising an antibody binding site, e.g., Fab,Fab', and F(ab')₂ fragments, or aggregates thereof.

The present invention provides means for determining cPSA in a bloodsample by measuring tPSA by immunoassay after rendering fPSA in theblood sample nondetectable. It will be evident to one of ordinary skillin the art that a variety of immunoassay methods can be used to measuretPSA and that a variety of means can be employed to render fPSA in theblood sample nondetectable.

In general, tPSA immunoassay methods are either competitive ornoncompetitive. The former methods typically employ an immobilized orimmobilizable antibody to PSA (anti-PSA) and a labeled form of PSA.Sample PSA and labeled PSA compete for binding to anti-PSA. Afterseparation of the resulting labeled PSA that has become bound toanti-PSA (bound fraction) from that which has remained unbound (unboundfraction), the amount of the label in either bound or unbound fractionis measured and can be related to the amount of PSA in the test samplein any conventional manner, e.g., by comparison to a standard curve.

Non-competitive methods are more commonly used for the determination oftPSA, with the most common method being the two-site immunometric assaymethod (sometimes referred to as the "sandwich" method). In immunometricassays, two anti-PSA antibodies are employed. One of the anti-PSAantibodies is labeled (sometimes referred to as the "detectionantibody") and the other is immobilized or immobilizable (sometimesreferred to as the "capture antibody"). As is known in the art, thecapture and detection antibodies can be contacted simultaneously orsequentially with the test sample. Sequential methods can beaccomplished by incubating the capture antibody with the sample, andadding the labeled antibody a predetermined time thereafter (sometimesreferred to as the "forward" method); or the detection antibody can beincubated with the sample first and then the labeled antibody added(sometimes referred to as the "reverse" method). After the necessaryincubation(s) have occurred, to complete the assay, the capture antibodyis separated from the liquid test mixture, and the label is measured inat least a portion of at least one of the separated capture antibodyphase or the remainder of the liquid test mixture, normally the formersince it comprises PSA bound by ("sandwiched" between) the capture anddetection antibodies.

In typical two-site immunometric assays for PSA, one or both of thecapture and detection antibodies are monoclonal antibodies. The labelused in the detection antibody can be selected from any of those knownconventionally in the art. Commonly, the label is an enzyme or achemiluminescent moiety, but can also be a radioactive isotope, afluorophor, a detectable ligand (e.g., detectable by a secondary bindingby a labeled binding partner for the ligand), and the like. Theimportant property of the capture antibody is that it provides a meansfor being separated from the remainder of the test mixture. Accordingly,as is understood in the art, the capture antibody can be introduced tothe assay in an already immobilized or insoluble form, or can be in aimmobilizable form, that is, a form which enables immobilization to beaccomplished subsequent to introduction of the capture antibody to theassay. Examples of immobilized capture antibody are antibody covalentlyor noncovalently attached to a solid phase such as a magnetic particle,a latex particle, a microtiter plate well, a bead, a cuvette, or otherreaction vessel. An example of an immobilizable capture antibody isantibody which has been chemically modified with a ligand moiety, e.g.,a hapten, biotin, or the like, and which can thus be subsequentlyimmobilized by contact with an immobilized (as described above fordirectly immobilized capture antibody) form of a binding partner for theligand, e.g., an antibody, avidin, or the like.

The above-described immunoassay methods and formats are intended to beexemplary and are not limiting since, in general, it will be understoodthat any immunoassay method or format can be used in the presentinvention.

It will also be understood that the means employed for rendering samplefPSA nondetectable in a particular immunoassay can vary widely. In oneaspect, such means can involve isolation or separation of sample fPSAfrom the remainder of the blood sample in which the immunoassay isperformed. Such separation can result in physical separation of the FPSAfraction from the liquid test mixture or can result in isolation orsequestration of fPSA in situ in the test mixture. By way of example,fPSA can be separated and rendered nondetectable by passing the testsample through a column or other matrix of material which selectivelyremoves fPSA such as by ion exchange adsorption, molecular sievefiltration, affity binding, or the like, or by contacting the testsample with an immobilized or immobilizable form of fpSA-specificantibody, such as anti-fPSA fixed to a magnetic or latex particle.

In another aspect, FPSA is rendered nondetectable by immunoassay bytreatment of the test sample with physical, chemical (includingbiochemical), or other means which convert or modify the relevantantigenic determinant(s) sufficiently to render fPSA substantiallyincapable of binding with antibody involved in the PSA immunoassay. ThePSA immunoassay can then be performed directly in the resulting testmixture. By way of example, such treatment can involve differentialdenaturation of fPSA and cPSA such as by heating or cooling; addition ofa chemical denaturant which denatures fPSA antigenic determinants(E-epitope), while being ineffective against cPSA determinants which areprotected by complexation, such as proteases specific for peptidesunique for the E-epitope region and the like; addition of a biochemicalagent which binds or otherwise blocks the E-epitope region such asprotein or lipid binders, substrate mimics (e.g., peptides whichresemble a normal substrate recognized by an enzymatic site in theE-epitope region, but which bind without subsequent enzymatic cleavageor reaction); and the like. The above means for accomplishing thedesired immunological inactivation of fPSA are not intended to beexhaustive and other effective methods will be evident to the ordinaryworker in the field.

A particularly unique method for determining cPSA provided by thepresent invention involves an ingenious modification of a conventionaltwo-site immunometric assay. In this new method, one of the capture anddetection antibodies is selected to be capable of binding tPSA (that is,it binds to an epitope that is available on both fPSA and cPSA), butsubstantially incapable of binding to PSA when PSA is bound by afPSA-specific antibody (i.e., an anti-E antibody). This unique antibodyis referred to herein as MM1. Thus, by adding anti-E as a thirdantibody, FPSA which becomes bound by anti-E is rendered substantiallyincapable of binding to MM1, and thus substantially incapable of beingdetected in a two-site immunometric assay based on MM1.

In this particularly preferred method, the second antibody (the MM1antibody) and the third antibody (the anti-E antibody) independently areeach preferably a monospecific antibody (e.g., a monoclonal antibody ora polyclonal antibody obtained by a conventional antiserum method whichhas been prepared such that the antibody fraction consists essentiallyonly of antibodies that bind to the specific epitope of interest), andmost preferably is a monoclonal antibody. Moreover, the anti-E antibodycan, if desired, comprise more than one antibody, e.g., more than onemonoclonal antibody, in order to obtain the desired inhibition of MM1.It will be further understood that the desired degree of inhibition ofbinding of the MM1 antibody to FPSA caused by the binding of the anti-Eantibody (or antibodies) will normally be greater than about 90%, moreusually greater than about 95%, and most preferably greater than about99%.

Particularly preferred monoclonal MM1 antibodies can be prepared in anumber of ways. Principally, the monoclonal antibody will be prepared byapplying conventional somatic cell hybridization techniques using ascreening method for selection of hybridoma cell lines which results inisolation of hybridomas which produce a monoclonal antibody having thedefined binding properties of MM1. The strategy for such screening is toselect antibodies that block the binding of other antibodies directed toepitopes accessible on FPSA but not cPSA, e.g., E-epitope, butthemselves have substantially equivalent binding to fPSA and cPSA.

Somatic cell hybridization is now a well-known methodology and can beapplied to the present invention in all of its variations as appropriateand desired. In general, one prepares a population of hybridomas byfusion of myeloma cells with lymphocyte cells taken from an animal thathas been immunized against the analyte. Immunization of the host animal,as used herein, implies that the animal's immune system has beenchallenged to produce antibodies that will bind to one or more epitopeson the analyte of interest. It will be evident to the skilled worker inthe art that such result can be obtained in any number of ways,including, without limitation, administration of the native analyte,synthetic peptide inimunogen, tranfectant cells which express epitopesof the analyte on their surface, or the like, to the bloodstream of thehost animal. Similarly, production and harvesting of monoclonalantibodies from cloned hybridoma cell lines are within the ordinaryskill in the art, and in general any known method can be used inpracticing the present invention.

As described above, the principal criteria for screening of hybridomasto produce a monoclonal antibody having MM1 characteristics are that itproduce a monoclonal antibody which (i) binds substantially equivalentlyto fPSA and cPSA, but (ii) is substantially incapable of binding to PSAwhen PSA is bound by a fPSA-specific antibody (an anti-E antibody).Alternatively, but less preferably, the screening criteria can be thatit produce a monoclonal antibody which (i) binds substantiallyequivalently to fPSA and cPSA, but (ii) upon becoming bound to PSA,renders PSA substantially incapable of binding to antibodies that arespecific for fPSA, that is, antibodies that can bind fPSA, but not cPSA.The mechanism by which the MM1 monoclonal antibody of the presentinvention operates to provide the above result is not clearlyunderstood; however, it is speculated that the binding of the monoclonalantibody to FPSA (i.e., anti-E antibody) blocks, masks, obscures, oralters the epitope(s) that are available on both fPSA and cPSA forbinding by an antibody or antibodies directed to such epitope(s).Representative of the MM1 monoclonal antibody employed in the presentinvention are the MM1 antibody used in the Bayer Immuno 1™ PSA Assay(Bayer Corporation, Tarrytown, N.Y., USA); the monoclonal antibodiesproduced by hybridoma cell lines 346.7.4 and 346.7.26 deposited by theassignee of the present application (Bayer Corporation) with theAmerican Type Culture Collection, Rockville, Md., USA, on Apr. 10, 1997,and assigned deposit numbers HB-12338 and HB-12337, respectively; andmonoclonal antibodies that bind to substantially the same epitope as anyof the aforesaid antibodies.

The reagents and other assay components necessary for practice of theabovedescribed particularly preferred method for determining cPSA areconveniently provided in the form of a test kit, that is, a packagedcollection or combination as appropriate for the needs of the user andany analytical instrumentation involved. Minimally, the test kit willcomprise the particularly characterized capture and detection antibodiesand one or more anti-E antibodies.

cPSA blood values in male patients have now been found to providesubstantial clinical significance in comparison to the prior art tPSAvalues and fPSA/tPSA ratio values. Specifically, an initial study wasconducted using serum samples from 216 patients including 53 patientswith CaP, 75 patients with BPH, and 88 healthy male controls over theage of 50 years (infra, the data presented in FIG. 4A). In this initialstudy, the upper limit of normal of the cPSA assay was established toprovide equivalent sensitivity for detection of CaP as compared withmeasurement of tpSA (85% v. 88%, respectively). For all patients tested,the specificity in the normal and BPH populations was also comparablefor cPSA compared to the fPSA/tPSA ratio. These findings were confirmedin a subsequent study using serum samples from 300 biopsied patientsfrom a urology referral population including 75 patients with CaP, and225 patients found to be free of CaP by biopsy (infra, the datapresented in FIG. 3B). The finding that the sensitivity and specificityof the tPSA assay used in conjunction with a fPSA/tPSA ratio isequivalent to that of the cPSA alone also held true when the patientpopulation was stratified into the diagnostic gray zone. The preciserange of the diagnostic gray zone has not been defined, but at allranges compared in this study, the sensitivity and specificity of thecPSA assay was comparable to that obtained using both total and free PSAassays. These data demonstrate that a single test, cPSA, can detectprostate cancer as efficiently as total PSA, and, in addition, has theimproved specificity that has been shown to be obtainable using twoassays, FPSA and tPSA.

In the studies referred to above, the upper limit of normal (sometimesreferred to as the cut-off value) selected for the cPSA assay data was3.75 ng/mL (expressed as an equivalent PSA concentration). This upperlimit of normal value was selected in order to achieve a sensitivity forCaP detection in the group of men with histologically confirmed cancersubstantially similar to that provided using a 4.0 ng/mL cut-off withthe tPSA assay (85% compared to 88% in the initial study and 81%compared to 83% in the subsequent study). It will of course beunderstood in the art that as larger sample populations are tested, theoptimum upper limit of normal for cPSA values may shift to some degree,however, it would be anticipated that such optimum upper limit of normalwill, in any event, fall approximately between 3-4 ng/mL (equivalent toapproximately 9-12 ng/mL PSA-ACT). Selection of an upper limit of normalabove 4 ng/mL would generally be understood to result in a clinicallyunacceptable level of sensitivity, while selection of an upper limit ofnormal below 3 ng/mL might be considered by some clinicians to affordincreased sensitivity with an acceptable loss in specificity. However,it will be recognized that for any given level of sensitivity, the cPSAmethod of the present invention will provide, in a single assay result,a significantly improved level of specificity as compared withconventional tPSA methods, and an equivalent or improved level ofspecificity as compared with recently published methods based on theratioing of two assay results, e.g., fPSA/tPSA. It is furthercontemplated that detection of prostate cancer in an asymptomatic malepatient will be enhanced by serial measurement of cPSA over time as hasbeen shown for serial tPSA measurements (29).

In addition to the above-discussed use in the detection of prostatecancer, the measurement of cPSA will be useful in the monitoring of thecourse of disease in patients who have been diagnosed with prostatecancer, particularly after having received first line therapy forprostate cancer. Longitudinal monitoring of such patients by measurementof TPSA has been proven to be useful in the early detection of recurrentprostate cancer. cPSA is understood to be the cancer-specific form ofPSA and is the form that would be expected to increase in serum ascryptic cancer cells establish distant metastic sites and grow.Accordingly, changes in cPSA blood levels over time will correlate withchanges in disease status, and particularly, increasing cPSA bloodlevels after therapy will indicate recurrence of disease.

Furthermore, since it is understood that cPSA is comprised primarily ofPSA-ACT, the clinical significance and advantages of cPSA measurementsextend to PSA-ACT measurements as well (as demonstrated in the studywhich produced the data presented in FIG. 6). In principle, immunoassaymethods for the determination of PSA-ACT that would be most amenable forperformance on instrumentation currently available would be two-siteimmunometric assays using anti-PSA antibody in combination with ananti-ACT antibody or an antibody that is specific for PSA-ACT complex.One method for attaining such latter antibody is by monoclonal selectionof an antibody which is directed to a conformational epitope on thePSA-ACT complex, e.g., at or near the point on the surface of thecomplex where the ACT and PSA components meet. Presently, however, suchmethods are not well developed and/or suffer from analytical performanceproblems, and accordingly, until further improvements are forthcoming,measurement of PSA-ACT will require more cumbersome techniques. Forexample, as shown by Leinonen et al., PSA-ACT complexes may be separatedby gel filtration (molecular sieve) chromatography and measured inassays which detect either tPSA or are specific for PSA-ACT (15).

The present invention will now be illustrated, but is not intended to belimited by, the following examples.

EXAMPLES

Materials. Anti-PSA antibodies used in these studies include MM1, amonoclonal antibody which recognizes an epitope expressed on free PSAand PSA complexed with proteinase inhibitors. The antibody was producedin mouse ascites fluids and purified by protein A affinitychromatography using standard procedures. MP2 is a polyclonal anti-PSAantibody which was produced in goats and purified by affinitychromatography on immobilized PSA. PSA 19, PSA 20, PSA 30 (CanAgDiagnostics AB, Gothenburg, Sweden) and ME2 (Biospacific, Emeryville,CA, USA) are monoclonal antibodies which recognize the E-epitope of PSA.ACT 53 (CanAg Diagnostics) is an ACT-specific monoclonal antibody. Freeprostate-specific antigen (Scripps Laboratories, San Diego, Calif., USA)was purified from human seminal fluid with 98% purity by sodium dodecylsulfate-polyacrylamide gel electrophoresis and was stored in a buffercontaining 10 mM Tris, 0.1% sodium azide, pH 8.0. PSA-ACT (ScrippsLaboratories, San Diego, Calif., USA) showed >96% purity by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, and was stored in abuffer containing 10 mM sodium acetate, 150 mM sodium chloride, and 0.1% sodium azide, pH 5.6.

The Bayer Immuno 1™ PSA Assay. The Bayer Immuno 1 total PSA (tPSA) assayis a sandwich assay which uses a monoclonal antibody for capture and apolyclonal antibody for detection of PSA. The monoclonal anti-PSAantibody (MM1) is conjugated to fluorescein (R1) and theaffmity-affinity-purified polyclonal antibody (MP2) is conjugated toalkaline phosphatase (R2). The antibodies are diluted to 1.5 μg/ml forR1 and 6.15 μg/ml for R2 in a buffer containing 100 mM Tris-HCl, pH 7.4,and 5% heat-inactivated normal goat serum (Biocell Laboratories, Carson,Calif., USA). A 65 μl volume of each of the two antibodies are incubatedwith 20 μl of the test specimen in a reaction cuvette for 20 min at 37°C., and the resulting immunocomplex (R1-PSA-R2) is captured by theaddition of magnetic particles coated with monoclonal anti-fluoresceinantibodies (20 μL). After a wash step to remove excess reagents andsample components, 300 μl of 23 mM p-nitrophenyl phosphate is added. Therate of color formation is monitored by absorbance measurements at 405or 450 nm and the rate of color formation is directly proportional tothe concentration of PSA in the test specimen. Further details areprovided in J. Clin. Lab. Anal. (1996), 10:155-159. Calibration of theBayer Immuno 1 Analyzer is performed using the Bayer Immuno 1 SETpoints® PSA calibrators, prepared from free PSA at concentrations of 0,2, 10, 25, 50 and 100 ng/mL. A cubic-through-zero fitting algorithm isused to generate a standard calibration curve.

The Bayer Immuno 1 Free PSA Assay. The protocol used for the BayerImmuno 1 total PSA Assay described above was adapted for measurement offree PSA by the substitution of a monoclonal antibody specific for freePSA (PSA 19, CanAg) conjugated to fluorescein as the Ri captureantibody. The monoclonal anti-free PSA R1 was used with the samepolyclonal anti-PSA alkaline phosphatase conjugate (R2) as that used inthe total PSA Assay. The R1 conjugate was diluted to 2.5 μg/mL and theR2 was used at 6.15 μg/mL. Other conditions were similar to those usedin the tPSA Assay except that the sample volume was 35 μl per test andthe volume of magnetic particles added was 15 μL per test.

The Bayer Immuno 1 PSA-ACT Method. The Bayer Immuno 1 PSA-ACT assayformat is the same as that of the Bayer Immuno 1 tPSA Assay except forthe following changes: (1) a monoclonal antibody specific for ACT, ACT53, is conjugated to alkaline phosphatase and used for detection at 2μg/mL; (2) PSA-ACT is used as the calibrator and control antigen withthe 50 mM MES buffer, 6% BSA, pH 5.8; and (3) a two-wash protocol isused such that antigen is first incubated with capture antibody, theresulting complexed is washed to remove unbound antigen and other serumcomponents, and then the detection antibody is added.

Results

Selection and Optimization of Specific Antibodies for Inhibition of FreePSA Immunoreactivity in the Total PSA Assay. The present invention isbased on the observation that the total PSA Assay can be made specificfor PSA-protease inhibitor complexes by the addition of an antibody tothe E epitope of PSA. Four monoclonal antibodies, PSA 19, PSA 20, PSA30, and ME2, specific for the E-epitope on the PSA molecule, were testedfor their ability to decrease reactivity of free PSA in the total PSAAssay. The calibrators used in the total PSA Assay were prepared using100 % free PSA purified from seminal fluid. Anti-E antibodies, PSA 19,PSA 20 and PSA 30, were added to the 50 ng/ml PSA calibrator atconcentrations of 0, 10, 25, 50, 100, and 200 μg/mL. After incubation atroom temperature for 30 to 60 min these mixtures were run as unknownsamples using the total PSA assay and the recovery of PSA wasdetermined. As shown in FIG. 1A, each of the three monoclonalantibodies, PSA 19, PSA 20, and PSA 30 showed significant inhibition offree PSA reactivity in the total PSA Assay. This decreasedimmunoreactivity of free PSA was concentration dependent for each of theantibodies but only PSA 20 approached saturation. Of these three anti-Eantibodies, PSA 20 gave the greatest decrease in signal for free PSA.

In a separate experiment, PSA 20 and ME2 were compared for their abilityto inhibit the binding of free PSA in the tPSA assay. Monoclonalantibodies were added to the 10 ng/ml calibrator at concentrationsranging from 0 to 400 μg/ml. As can be seen in FIG. 1B, the ME2 MAbinhibits the binding of free PSA in the total PSA quantitatively andreaches saturation at a concentration of less than 6.125 μg/mL. Thesedata demonstrate that several E-epitope antibodies have the ability toinhibit the binding of the MM1 antibody to free PSA. However, the ME2MAb inhibits free PSA binding in the total PSA assay at a much lowerconcentration and to a greater extent than other E-epitope antibodies.This inhibition could be due to a higher affinity of the ME2 antibodyfor the E-epitope. Alternatively, the E-epitope may represent acollection of epitopes with different fine epitope specificities.

Measurement of Complexed PSA on the Bayer Immuno 1 Analyzer. Theaddition of MAbs PSA 20 and ME2 to the total PSA Assay eliminates mostof the immunoreactivity associated with free PSA. To demonstratequantitative measurement of complexed PSA, mixtures with variousproportions of free and ACT-complexed PSA were prepared at a total PSAconcentration of approximately 11 ng/mL. The mixtures contained ratiosof free:complexed PSA of 100:0, 80:20, 50:50, 20:80, and 0:100. Thesemixtures were measured using three immunoassay formats on the BayerImmuno 1 Analyzer: the commercial assay for total PSA (tPSA), the BayerImmuno 1 free PSA Assay (fPSA), and the Bayer Immuno 1 complexed PSAAssay (cPSA). The Bayer Immuno 1 complexed PSA assay was identical tothe tPSA assay except that for results shown in FIG. 2A, PSA 20 MAb wasadded to each sample at a final concentration of 300 μg/ml and theMM1-fluorescein conjugate was reduced from 1.5 μg/ml to 0.5 μg/ml. Forthe experiment shown in FIG. 2B, the MAb ME2 was added to each sample ata final concentration of 25 μg/mL and the MM1-fluorescein conjugate wasagain used at 0.5 μg/mL. For the measurement of total PSA and free PSA,the Bayer Immuno 1 Analyzer was calibrated with the Bayer Immuno 1 SETpoint PSA calibrator set which is used commercially for the Bayer Immuno1 total PSA Assay. To measure complexed PSA (cPSA), calibrators in therange of 0-100 ng/mL were prepared using PSA complexed with ACT in 50 mMMES, 6% BSA, pH 5.8.

The addition of MAb PSA 20 to the total PSA Assay provides a method withalmost quantitative reactivity with complexed PSA (FIG. 2A). Theresponse for the various mixtures in the cPSA assay was linear, and themeasured concentration of total PSA and complexed PSA gave consistentrecoveries of approximately 10 ng/mL for all samples tested, asexpected. Similarly, MAb ME2 provides a method with quantitativereactivity with complexed PSA over the complete range of proportions offree and complexed PSA (FIG. 2B). In addition, the complexed PSA assaywith MAb ME2 uses a significantly lower concentration of the ME2 MAb (25μg/mL) compared with that required for the PSA 20 MAb (300 μg/mL). Thesedata demonstrate that three antibodies which react with differentepitopes on the PSA molecule (MM1, MP2, and either PSA 20 or ME2), canbe used in combination to produce a method which accurately measurescomplexed PSA.

Automation of the cPSA Assay. Pretreatment of patient samples with MAbto the E-epitope is not practical for application in the clinicallaboratory environment. Accurate dispensing of MAb into the sample isdifficult and time consuming, and leads to an unacceptably highprobability of inaccuracy in the result. Methods for full automation ofthe cPSA Assay were therefore developed.

Automated MAb PSA 20 Methods. In assay format 1, MAb PSA 20 was added tothe R1, MM1-fluorescein, reagent at a concentration of 500 μg/mL, andthe assay was run as for the tPSA method using PSA-ACT for calibration.This assay takes 38 minutes for completion. In assay format 2, thesample is pretreated with MAb PSA 20 onboard. In this format, PSA 20antibody was added to the reaction cuvette together with the patientsample and incubated for 50 minutes. MM1-fluorescein at a concentrationof 0.5 μg/mL, MP2-ALP at a concentration of 6.15 μg/mL, and magneticparticles coated with anti-fluorescein antibodies were then added andincubated for an additional 28 minutes. After washing away excessreagents and unreacted serum, substrate was added and color formationwas monitored in the same manner as for the tPSA Assay. Samplescontaining free PSA over a concentration range from 2 ng/ml-25 ng/mlwere used. Results in FIG. 3 show that the signal with free PSA can beeffectively reduced to very low levels using either of these approaches.These results suggest that this method can be fully automated on theBayer Immuno 1 Analyzer.

Automated MAb ME2 Method. ME2 MAb inhibits the binding of the MM1 MAb tofPSA in the tPSA assay at a significantly lower concentration and to agreater degree than PSA 20 MAb. Additionally, assay format 2 takes alonger time and needs two reagent cassettes. Therefore, ME2 MAb wasselected to be used as a third antibody for automation of the cPSA assayby addition into the tPSA assay in two ways--ME2 at a concentration of50 and 100 ng/mL was added into either the reagent 1 (R1) or the reagent2 (R2). Results showed that fPSA reactivity was inhibited 97% and 98%when ME2 was added into RI and R2, respectively. Based on these data, itwas determined that the cPSA assay would be formulated using ME2 MAb inthe R2 reagent at a final concentration of 100 μg/mL.

Measurement of Complexed PSA in Serum.

Pilot Study.--Serum samples from 53 patients with prostate cancer, 75patients with BPH, and 88 samples from healthy age-matched controlsubjects were analyzed using the three assays: tPSA, fPSA, and cPSA.Samples tested in the cPSA assay were pretreated with 25 μg/ml ME2antibody, and samples tested in the fPSA and TPSA assays were untreated.The assays were calibrated using either free PSA or PSA-ACT complexes asdescribed above. The results of this testing are shown in FIG. 4A. Theupper limit of normal value of 3.75 ng/mL (expressed as an equivalentPSA concentration) was selected in order to achieve a sensitivity forCaP detection in the group of men with histologically confirmed cancersubstantially similar to that provided using a 4.0 ng/mL cut-off withthe TPSA assay (85% compared to 88%). With this upper limit of normal,the specificity in the normal and BPH populations tested in this studywas also comparable for cPSA compared to a two-step test in which apositive TPSA result was followed by running a fPSA assay andcalculating the fPSA/tPSA ratio. The finding that the sensitivity andspecificity of the TPSA assay used in conjunction with a fPSA/tPSA ratiois equivalent to that of the cPSA alone, also held true when the patientpopulation was stratified into the diagnostic gray zone. The preciserange of the diagnostic gray zone has not been defined, but at allranges compared in this study, the sensitivity and specificity of thecPSA assay was comparable to that obtained using both total and free PSAassays. These data demonstrate that a single test, cPSA, can detectprostate cancer as efficiently as total PSA, and, in addition, has theimproved specificity that has been shown to be obtainable using twoassays, fPSA and tPSA.

Clinical Study.--Serum samples from 300 biopsied patients (75 withconfirmed prostate cancer) were analyzed at the Seattle VA Hospital,Seattle, Wash., USA using the three assays: tPSA (using the HybritechTandem® PSA Assay, San Diego, Calif., USA), fPSA (using the HybritechTandem® free PSA Assay), and cPSA (using the automated MAb ME2 methoddescribed above). The assays were calibrated using either free PSA orPSA-ACT complexes as described above. The results of this testing areshown in table form in FIG. 4B and as a regression analysis in FIG. 4C.The upper limit of normal value of 3.75 ng/mL (expressed as anequivalent PSA concentration) was again selected in order to achieve asensitivity for CaP detection in the group of men with histologicallyconfirmed cancer substantially similar to that provided using a 4.0ng/mL cut-off with the tPSA assay (81 % compared to 83%). With thisupper limit of normal, the specificity in the normal and BPH populationstested in this study was also comparable for cPSA compared to thetwo-step test (supra, tPSA+fPSA/tPSA). In FIG. 4C, the small circle datapoints appearing in the lower right quadrant of the graph representthose non-cancer patients (34 out of 117, or 29%) who would have avoidedthe risk, discomfort and expense of biopsy had their cPSA, rather thantheir tPSA, value been used as the basis of this medical decision. Also,as in the pilot study, the finding that the sensitivity and specificityof the tPSA assay used in conjunction with a fPSA/tPSA ratio isequivalent to that of the cPSA alone, held true when the patientpopulation was stratified into the diagnostic gray zone (supra).

The data from the independent pilot and clinical studies demonstratethat a single test, cPSA, can detect prostate cancer as efficiently astotal PSA, and, in addition, has the improved specificity that has beenshown to be obtainable using two assays, FPSA and tPSA.

Correlation Between CPSA and PSA-ACT. To determine what species ofcomplexed PSA are measured in the cPSA assay, an assay was developed tomeasure PSA complexed with ACT. This species of complexed PSA has beenreported to represent the predominant form of complexed PSA in serum.Previous attempts to measure PSA-ACT using manual methods have met withtechnical difficulties as discussed above. Accordingly, an automatedimmunoassay was developed for measuring PSA-ACT complexes on the BayerImmuno 1™ system. The same population of patients described above in thepilot study were tested using the automated assay for PSA-ACT. Resultsare shown in FIGS. 6A-6F where results obtained using the automatedPSA-ACT assay are regressed against results obtained using the automatedcPSA assay. For each patient population, i.e., normals, prostate cancer,and benign prostate disease (BPH), regression analysis was done for allpatient samples, and over the range containing the majority of patienttest results. This was done to eliminate bias in the regression analysisdue to a small number of high values. In any event, the slopes of theregressions ranged from 0.93-0.98. These data suggest that approximately93-98% of the substances measured by the cPSA assay is PSA-ACT. Thebiochemical nature of the remaining 2-7% of cPSA is not known at thistime.

Specificity of PSA Assays at Selected Sensitivities. The upper limit ofnormal used for the cPSA assay was determined to give similarsensitivity as the tPSA assay. Using this cut-off value, it wasdemonstrated that the cPSA assay provides improved specificity overmethods currently used in medical practice, i.e., tpSA. The specificityof the cPSA assay was also measured using different values for the upperlimit of normal. All results derived from the clinical study describedabove were used in a Receiver Operator Characteristic (ROC) analysis.Specificity was then determined from the ROC analysis at varying levelsof sensitivity ranging from 80-100%. Sensitivities of less than 80% havelittle medical value since diagnostic methods in current practiceprovide at least this level of sensitivity. Results shown in FIG. 7demonstrate that at all levels of sensitivity, the cPSA assay providesadditional sensitivity over tPSA and approximately equivalent orslightly better specificity than the use of two assays, tPSA and fPSA.In addition, the improvement in specificity holds true even when thepatient samples are stratified in the diagnostic gray zone. Theseresults further demonstrate that the upper limit of normal for the cPSAassay can be chosen over a broad range, depending on the desired levelof sensitivity and specificity, but at all values for the upper limit ofnormal ranging between about 3-4 ng/mL, cPSA gives improved specificityover tPSA, and approximately equivalent specificity as the fPSA/tPSAratio.

The present invention has been particularly described and exemplifiedabove. Clearly, many other variations and modifications of the inventioncan be made without departing from the spirit and scope hereof.

BIBLIOGRAPHY

1. Wang M C, Valenzuela L A, Murphy G P, Chu T M. Purification of ahuman prostate specific antigen. Invest Urol 1979; 17:159-63

2. Watt W K, Lee P J, Timkulu T M, Chan W P, Loor R. Humanprostate-specific antigen: structure and functional similarity withserine proteases. Proc Natl Acad Sci USA 1986; 83:3166-70.

3. Akiyama K, Nakamura T, Iwanaga S, Hara M. The chymotrypsin-likeactivity of human prostate-specific antigen, r-seminoprotein. FEBSLetters 1987; 225:168-72.

4. Christensson A, Laurel C B, Lilja H. Enzymatic activity ofprostatic-specific antigen and its reactions with extracellular serineproteinase inhibitors. Eur J Biochem 1990; 194:755-763.

5. Lilja H. A kallikrein-like serine protease in prostatic fluid cleavesthe predominant seminal vesicle protein. J Clin Invest 1985;76:1899-903.

6. Lilja H, Abrahamsson P A. Three predominant proteins secreted by thehuman prostate gland. Prostate 1980; 12:29-38.

7. Papsidero L D, Wang M C, Valenzuela I A, Murphy G P, Chu T M. Aprostate antigen in sera of prostate cancer patients. Cancer Res 1980;40:2428-2432.

8. Lange P H. Prostate specific antigen in diagnosis and management ofprostate cancer. Supplement to Urology 1990; XXXVI:25-29.

9. Oesterling J E. Prostate specific antigen: a critical assessment ofthe most useful tumor marker for adenocarcinoma of the prostate. J Urol1991; 145:907-923.

10. Armbruster D A. Prostate-specific antigen: biochemistry, analyticalmethods and clinical application. Clin Chem 1993; 39:181-195.

11. American Cancer Society: Cancer facts & figures-1995. AmericanCancer Society, Inc., Atlanta, Ga. Page 11.

12. Catalona W J, Richie J P, Ahmann F R, Hudson M A, Scardino P T,Flanigan R C, deKernion J B, Ratliff T L, Kavoussi L R, Dalkin B L,Waters, W B, MacFarlane M T and Southwick PC. Comparison of digitalrectal examination and serum prostate specific antigen in the earlydetection of prostate cancer: results of a multicenter clinical trial of6,630 men. J Urol 1994; 151:1283-1290.

13. Stenman U H, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, AlfthanO. A complex between prostate-specific antigen and α121-antichymotrypsinis the major form of prostate-specific antigen in serum of patients withprostatic cancer: assay of the complex improves clinical sensitivity forcancer. Cancer Res 1991; 51:222-226.

14. Zhou A M, Tewari P C, Bluestein B L, Caldwell, G W, Larsen F L.Multiple forms of prostate-specific antigen in serum: differences inimmunorecognition by monoclonal and polyclonal assays. Clin Chem 1993;39:2483-91.

15. Leinonen J, Lovgren T, Vornanen T, Stenman U H. Double-labeltime-resolved immunofluorometric of prostate-specific antigen and itscomplex with α₁ -antichymotrypsin. Clin Chem 1993: 39:2098-2103.

16. Christensson A, Bjork T, Nilsson O, Nilsson O, Dahlen U, MatikainenM T, Cockett A T, Abrahamssion P A. Serum prostate specific antigencomplexed with α₁ -antichymotrypsin as an indicator of prostate cancer.J Urol 1993; 150:100-105.

17. Lilja H, Significance of different molecular forms of serum PSA. Thefree, noncomplexed forms of PSA versus that complexed to α₁-antichymotrypsin. Urol Clin North Am 1993; 20(4):681-686.

18. Lilja H, Christensson A. Dahlen U, Matikainen M-T, Nilsson 0,Pettersson K and Lovgren T: Prostate-specific antigen in human serumoccurs predominantly in complex with alpha-antichymotrypsin. Clin Chem1991; 37:1618-1625.

19. Bjork T, Hulkko S, Bjartell A, Santagnese A D, Abrahamnsson P-A,Lilja H. Alpha₁ -antichymotrypsin production in PSA-producing cells iscommon in prostate cancer but rare in benign prostatic hyperplasia.Urology 1994; 43:427-434.

20. Pettersson K, Piironen T, Seppala M, Liukkonen L, Christensson A,Matikainen M-T, Suonpaa M, Lovgren T, Lilja H. Free and complexedprostate-specific antigen (PSA): in vitro stability, epitope map, anddevelopment of immunofluorometric assays for specific and sensitivedetection of free PSA and PSA-α₁ -antichymotrypsin complex. Clin Chem1995; 41:1480-1488.

21. W u J T, Wilson L, Zhang P, Meiklde A W, Stephenson R. Correlationof serum concentrations of PSA-ACT complex with total PSA in random andserial specimens from patients with BPH and prostate cancer. J Clin LabAnal 1995 9:15-24.

22. Mitrunen K, Pettersson K, Piironen T, Bjork T, Lilja H, Lovgren T.(1995) Dual label one-step immunoassay for simultaneous measurement offree and total prostate specific antigen concentrations and ratios inserum. Clin Chem 1995 41(8):1115-1120.

23. Catalona W J, Smith D S, Wolfert R L, Wang T J, Rittenhouse H G,Ratliff T L, Nadler R B. Evaluation of percentage of free serumprostate-specific antigen to improve specificity of prostate cancerscreening. JAMA 1995 274(15): 1214-1220.

24. Prestigiacomo A F, Stamey T A. Clinical usefulness of free andcomplexed PSA. Scan J Clin Lab Invest 1995 55Supple221:32-34.

25. Wang T J, Hill T M, Sokoloff R L, Frankenne F. Rittenhouse H G,Wolfert R L. Dual monoclonal antibody immunoassay for freeprostate-specific antigen. Prostate 1996 28:10-16.

26. Jung K, Stephan C, Lein M, Henkne W, Schnorr D, Brux B,Schurenkamper P, Loening SA. Analytical performance and clinicalvalidity of two free prostatespecific antigen assays compared. Clin Chem1996 42(7):1026-1033.

27. Wang T J, Hill T, Sokoloff R, Frankenne F, Wolfert R, andRittenhouse H. Monoclonal antibody sandwich immunoassay to quantitatefree PSA in benign hyperplasia and prostate cancer. Poster presentationat 1994 ISOBM meeting.

28. Chan D, Kelley C A, Partin A W, Linton J, Wang T J, Sokoloff R L,Rittenhouse H G, and Wolfert R L. Clin Chem (1996) 42(6):S255.

29. Wang T J, Linton J, Payne J, Rittenhouse H G, Wolfert R L, Chan D W,Kelley C A and Partin A W. Clinical utility of a complexed PSAimmunoassay with a specific monoclonal antibody to PSA-ACT. J Urology(1997) 157(4) Suppl: 147.

30. Wang T J, Linton H J, Payne J, Liu R-S, Kuus-Reichel K, RittenhouseH G, Kelley C, Cox J, Chan D W, and Wolfert R L. Development ofmonoclonal antibodies specific for the PSA-ACT complex and theirincorporation into an immunoassay. Clin Chem (1997)43(6):S225.

31. Zhang P and W u, J T. Development of an immunoassay for the PSA-ACTcomplex in serum without interference of non-specific adsorption. ClinChem (1997) 43(6):S236.

32. Bjork T, Bjartell A, Abrahamsson P-A, Hulkko S, Di Sant'agnese A,Lilja H. Alpha₁ -antichymotrypsin production in PSA-producing cells iscommon in prostate cancer but rate in benign prostatic hyperplasia. Urol1994 43(4):427-434.

33. Carter H B, Pearson J D, Metter J, Brant L J, Chan D W, Andres R,Fozard J L and Walsh P C. Longitudinal evaluation of prostate-specificantigen levels in men with and without prostate disease. JAMA 1992267(16):2215.

What is claimed is:
 1. A method for determining complexed prostatespecific antigen (CPSA) in a blood sample, comprising the steps of:(a)treating the total amount of immunologically determinable PSA (tPSA) inthe blood sample to render free PSA (fPSA) substantially nondetectableby immunoassay, and (b) determining PSA in the treated blood sample byimmunoassay, whereby substantially only cPSA is detectable in saidassay.
 2. The method of claim 1 wherein step (a) is accomplished byseparating fPSA from the remainder of the blood sample and wherein step(b) is performed on said remainder of the blood sample.
 3. The method ofclaim 1 wherein step (a) is accomplished by addition to the blood sampleof an antibody to render fPSA substantially incapable of binding withantibody used in the immunoassay, and wherein step (b) is performed inthe test mixture produced by the addition of the antibody to the bloodsample.
 4. The method of claim 1 wherein said immunoassay is acompetitive immunoassay.
 5. The method of claim 1 wherein saidimmunoassay is a two-site immunometric assay.
 6. The method of claim 5wherein at least one of the two antibodies employed in the two-siteimmunometric assay is monoclonal.
 7. The method of claim 5 wherein bothof the two antibodies employed in the two-site immunometric assay aremonoclonal.
 8. A method for aiding in the differentiation of prostatecancer from benign prostate hypertrophy (BPH) in male human patients,comprising the steps of:(a) measuring the amount of complexed prostatespecific antigen (cPSA) in a blood sample obtained from such patient,and (b) determining if the patient's cPSA blood level is greater than anupper limit of normal having a value between about 3-4 ng/mL.
 9. Themethod of claim 8 wherein the upper limit of normal is about 3.75 ng/mL.10. A method for monitoring the course of disease in a male patientdiagnosed with prostate cancer, comprising the performance of a seriesof immunoassays over time to determine changes in the level of complexedprostate specific antigen (cPSA) in blood samples obtained from suchpatient, whereby changes in the cPSA blood level correlate with changesin disease status.
 11. A method for monitoring the course of disease ina patient who has been treated for prostate cancer, comprising theperformance of a series of immunoassays over time to determine changesin the level of complexed prostate specific antigen (cPSA) in bloodsamples obtained from such patient, whereby increases in blood cPSAlevels indicate recurrence of disease.
 12. A method for aiding in thedifferentiation of prostate cancer from benign prostate hypertrophy(BPH) in male human patients, comprising the steps of:(a) measuring theamount of complexed prostate specific antigen (cPSA) in a blood sampleobtained from such patient, and (b) determining if the patient's cPSAblood level is greater than an upper limit of normal having a valuebetween about 3-4 ng/mL; wherein cPSA is measured by the method of anyone of claims 1-7.